CN118667019A - Monoclonal antibody against human TIM-3 - Google Patents

Abstract

The present invention provides anti-human Tim-3 IgV domain-specific antibodies and fragments thereof, as well as methods of using such antibodies and/or fragments.

Description

Monoclonal antibody against human TIM-3

This application is a divisional application of a patent application with an application date of August 21, 2019, application number "201980037800.1", and invention name "Monoclonal Antibody Against Human TIM-3".

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/720,234, filed on August 21, 2018, the contents of which are each incorporated by reference in their entirety into this application.

Sequence Listing

This application contains a sequence listing submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on August 21, 2019 is named SequenceListing.txt and is 15.6KB in size.

Background of the Invention

The disclosures of all publications, patents, patent application publications, and books cited herein are hereby incorporated by reference in their entireties into this application to more fully describe the art to which this invention pertains.

T cell immunoglobulin and mucin-3 (Tim-3), also known as hepatitis A virus cellular receptor 2 (HAVCR2), is a type I transmembrane protein that is an inhibitory receptor expressed by IFN-g-producing T cells, Foxp3 ⁺ regulatory T cells, and innate immune cells such as macrophages and dendritic cells. Tim-3 inhibits immune responses by interacting with its ligand. Tim-3 contains an N-terminal immunoglobulin (IgV) domain, a mucin domain with O-linked glycosylation sites, a stalk domain with N-linked glycosylation sites between the mucin domain and the transmembrane domain, a transmembrane domain, and a cytoplasmic tail (Monney 2002). The investigators' laboratory has previously reported that the IgV domain of Tim-3 is a functional domain that is able to bind carbohydrates (Cao 2007). The IgV domain of Tim-3 contains two non-classical disulfide bonds and a unique CC'-FG cleft, a structural feature that has been identified in all Tim family proteins but not in other immunoglobulin superfamily members. There are also reports that adaptive resistance to PD-1 blockade therapy is associated with upregulation of other immune checkpoints, including Tim-3 (Koyama 2016). In addition to carbohydrates (Cao 2007; Wilker 2007), Tim-3 ligands have been reported to include Galectin-9 (Zhu 2005), phosphatidylserine (DeKruyff 2010), HMGB1 (high mobility group protein B1 (Chiba 2012) and LILRB2 (leukocyte immunoglobulin-like receptor B2) (PCT Publication No. WO16/111947).

SUMMARY OF THE INVENTION

The present invention provides an antibody or an antigen-binding fragment thereof that binds to the IgV domain of human Tim-3 (T cell immunoglobulin and mucin-3) or the IgV-mucin-stalk domain of human Tim-3, comprising:

a) a heavy chain comprising one or more heavy chain CDRs selected from:

GYSFTGYTIN (SEQ ID NO: 1) complementarity determining region (CDR) 1 (CDR1));

LFNPYNGGTT(SEQ ID NO:2)(CDR2);

ARRYYGYDAMDY(SEQ ID NO:3)(CDR3);

or

GFNIKDYYMH(SEQ ID NO:7)(CDR1);

WIDPENDNTIY(SEQ ID NO:8)(CDR2);

ARDFGYVAWLVY (SEQ ID NO:9) (CDR3); and

b) a light chain comprising one or more light chain CDRs selected from:

KSSQSVLYSSNQKNHLA(SEQ ID NO:4)(CDR1);

WASTRES (SEQ ID NO: 5) (CDR2);

HQYLSSYT(SEQ ID NO:6)(CDR3);

or

KASQNVDTAVA(SEQ ID NO:10)(CDR1);

SASNRYT(SEQ ID NO:11)(CDR2);

QQYSSYPT (SEQ ID NO: 12) (CDR3).

The present invention provides an antibody or an antigen-binding fragment thereof that binds to the IgV domain of human Tim-3 or the IgV-mucin-stalk domain of human Tim-3, comprising:

A heavy chain comprising the following heavy chain CDRs:

GYSFTGYTIN(SEQ ID NO:1)(CDR1);

LFNPYNGGTT(SEQ ID NO:2)(CDR2);

ARRYYGYDAMDY (SEQ ID NO: 3) (CDR3); and

A light chain comprising the following light chain CDRs:

KSSQSVLYSSNQKNHLA(SEQ ID NO:4)(CDR1);

WASTRES (SEQ ID NO: 5) (CDR2);

HQYLSSYT (SEQ ID NO:6) (CDR3).

The present invention provides an antibody or an antigen-binding fragment thereof that binds to the IgV domain of human Tim-3 or the IgV-mucin-stalk domain of human Tim-3, comprising:

A heavy chain comprising the following heavy chain CDRs:

GFNIKDYYMH(SEQ ID NO:7)(CDR1);

WIDPENDNTIY(SEQ ID NO:8)(CDR2);

ARDFGYVAWLVY (SEQ ID NO:9) (CDR3); and

A light chain comprising the following light chain CDRs:

KASQNVDTAVA(SEQ ID NO:10)(CDR1);

SASNRYT(SEQ ID NO:11)(CDR2);

QQYSSYPT (SEQ ID NO: 12) (CDR3).

The present invention provides an isolated antibody that binds to the IgV domain of human Tim-3 or the IgV-mucin-stalk domain of human Tim-3 with an affinity of 10.0 nM KD or stronger.

The present invention provides a nucleic acid encoding an antibody heavy chain comprising one or more heavy chain CDRs selected from the group consisting of: GYSFTGYTIN (SEQ ID NO: 1) (CDR1);

LFNPYNGGTT(SEQ ID NO:2)(CDR2);

ARRYYGYDAMDY(SEQ ID NO:3)(CDR3).

The present invention provides a nucleic acid encoding an antibody heavy chain comprising one or more heavy chain CDRs selected from the group consisting of: GFNIKDYYMH (SEQ ID NO: 7) (CDR1);

WIDPENDNTIY(SEQ ID NO:8)(CDR2);

ARDFGYVAWLVY(SEQ ID NO:9)(CDR3).

The present invention provides a nucleic acid encoding an antibody light chain comprising one or more light chain CDRs selected from the group consisting of: KSSQSVLYSSNQKNHLA (SEQ ID NO: 4) (CDR1);

WASTRES (SEQ ID NO: 5) (CDR2);

HQYLSSYT (SEQ ID NO:6) (CDR3).

The present invention provides a nucleic acid encoding an antibody light chain comprising one or more light chain CDRs selected from the group consisting of: KASQNVDTAVA (SEQ ID NO: 10) (CDR1);

SASNRYT (SEQ ID NO: 11) (CDR2)

QQYSSYPT (SEQ ID NO: 12) (CDR3).

The invention provides a host cell comprising one or more nucleic acids of the invention.

The invention provides an antibody or fragment thereof of the invention linked or conjugated to a therapeutic agent.

The present invention provides a method of inhibiting human Tim-3 in a subject, the method comprising administering the antibody or fragment thereof of the present invention in an amount effective to inhibit human Tim-3.

The present invention provides a method of inhibiting Tim-3-mediated T cell suppression in a subject, the method comprising administering the antibody or fragment thereof of the present invention in an amount effective to inhibit Tim-3-mediated T cell suppression.

The present invention provides a method of treating cancer in a subject, the method comprising administering an antibody or fragment thereof of the present invention in an amount effective to treat the cancer in the subject.

The present invention provides a method for detecting human Tim-3-positive cells in a subject, the method comprising administering an antibody or fragment thereof of the present invention conjugated to a detectable marker in an amount effective to label human Tim-3-positive cells, and then detecting human Tim-3-positive cells in the subject by detecting the presence of the marker in the subject.

The present invention provides an isolated nucleic acid molecule encoding an antibody or fragment thereof of the present invention. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is cDNA. In one embodiment, the nucleic acid is RNA. In some embodiments, the antigen-binding fragment of the antibody is a Tim-3 IgV domain binding fragment thereof. In some embodiments, the antigen-binding fragment of the antibody is a human Tim-3 IgV domain binding fragment thereof.

The present invention provides a vector encoding the nucleic acid molecule of the present invention. The present invention provides a host cell comprising the nucleic acid molecule of the present invention, or comprising the vector of the present invention.

The present invention provides a method for preparing an anti-Tim-3 IgV domain antibody or a Tim-3 IgV domain binding fragment thereof, the method comprising culturing a host cell under conditions enabling the host cell to produce the anti-Tim-3 IgV domain antibody or the Tim-3 IgV domain binding fragment thereof.

The present invention provides a pharmaceutical composition comprising the anti-Tim-3 IgV domain antibody or the Tim-3 IgV domain binding fragment thereof of the present invention, and a pharmaceutically acceptable excipient.

The present invention provides a method for reducing Tim-3 activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-Tim-3 IgV domain antibody or a Tim-3 IgV domain binding fragment thereof of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: ELISA analysis shows that 50B5 can bind to Tim-3-IgV protein and Tim-3 full-length extracellular region protein (IgV-mucin-stalk domain), but not to human IgG control protein.

Figure 2: FACS analysis shows that 50B5 binds to a cell line expressing human Tim-3, but not to a cell line expressing mouse Tim-3. 50B5 (open histogram) or mouse IgG1 isotype control (shaded histogram)

Figure 3: Kinetic parameters of 50B5 detected by surface plasmon resonance.

Figure 4A-4B: 4A: In the mixed lymphocyte reaction assay, divided CD4 T cells and divided CD8 T cells were found to express Tim-3 on day 4, but non-divided CD4 T cells and non-divided CD8 T cells did not express Tim-3. 4B: In the mixed lymphocyte reaction assay, mAb 50B5 was found to significantly enhance the production of IFN-g by CD4 T cells and CD8 T cells on day 4; N=8; **P<0.01.

Figure 5: FACS analysis showing binding of 15B4 to a cell line expressing human Tim-3. 15B4 (open histograms) or mouse IgG1 isotype control (shaded histograms)

Figure 6: Kinetic parameters of surface plasmon resonance detection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an antibody or an antigen-binding fragment thereof that binds to the IgV domain of human Tim-3 (T cell immunoglobulin and mucin-3) or the IgV-mucin-stalk domain of human Tim-3, comprising:

a) a heavy chain comprising one or more heavy chain CDRs selected from:

GYSFTGYTIN(SEQ ID NO:1)(CDR1);

LFNPYNGGTT(SEQ ID NO:2)(CDR2);

ARRYYGYDAMDY(SEQ ID NO:3)(CDR3);

or

GFNIKDYYMH(SEQ ID NO:7)(CDR1);

WIDPENDNTIY(SEQ ID NO:8)(CDR2);

ARDFGYVAWLVY (SEQ ID NO:9) (CDR3); and

b) a light chain comprising one or more light chain CDRs selected from:

KSSQSVLYSSNQKNHLA(SEQ ID NO:4)(CDR1);

WASTRES (SEQ ID NO: 5) (CDR2);

HQYLSSYT(SEQ ID NO:6)(CDR3);

or

KASQNVDTAVA(SEQ ID NO:10)(CDR1);

SASNRYT(SEQ ID NO:11)(CDR2);

QQYSSYPT (SEQ ID NO: 12) (CDR3).

The present invention provides an antibody or an antigen-binding fragment thereof that binds to the IgV domain of human Tim-3 or the IgV-mucin-stalk domain of human Tim-3, comprising:

A heavy chain comprising the following heavy chain CDRs:

GYSFTGYTIN(SEQ ID NO:1)(CDR1);

LFNPYNGGTT(SEQ ID NO:2)(CDR2);

ARRYYGYDAMDY (SEQ ID NO:3) (CDR3); and

A light chain comprising the following light chain CDRs:

KSSQSVLYSSNQKNHLA(SEQ ID NO:4)(CDR1);

WASTRES (SEQ ID NO: 5) (CDR2);

HQYLSSYT (SEQ ID NO:6) (CDR3).

The present invention provides an antibody or an antigen-binding fragment thereof that binds to the IgV domain of human Tim-3 or the IgV-mucin-stalk domain of human Tim-3, comprising:

A heavy chain comprising the following heavy chain CDRs:

GFNIKDYYMH(SEQ ID NO:7)(CDR1);

WIDPENDNTIY(SEQ ID NO:8)(CDR2);

ARDFGYVAWLVY (SEQ ID NO:9) (CDR3); and

A light chain comprising the following light chain CDRs:

KASQNVDTAVA(SEQ ID NO:10)(CDR1);

SASNRYT(SEQ ID NO:11)(CDR2);

QQYSSYPT (SEQ ID NO: 12) (CDR3).

In some embodiments, the framework regions of the light and heavy chains are human framework regions, or are at least 85% identical to human framework regions.

In some embodiments, the framework regions of the light chain and heavy chain are human framework regions. The present invention provides an isolated antibody or antigen-binding fragment thereof, which binds to the IgV domain of human Tim-3 or the IgV-mucin-stalk domain of human Tim-3 with an affinity of 10.0 nM KD or stronger.

In some embodiments, the isolated antibody binds to the IgV domain of human Tim-3 or the IgV-mucin-stalk domain of human Tim-3 with an affinity of 1.0 nM KD or stronger. In some embodiments, the isolated antibody binds to the IgV domain of human Tim-3 or the IgV-mucin-stalk domain of human Tim-3 with an affinity of 0.5 nM KD or stronger.

In some embodiments, the isolated antibody does not bind to murine Tim-3.

In some embodiments, the isolated antibody inhibits binding of human Tim-3 to phosphatidylserine expressed on dexamethasone-treated Jurkat T cells.

In some embodiments, the isolated antibody or antigen-binding fragment thereof has a human Fc region sequence.

In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising one or more heavy chain CDRs selected from:

GYSFTGYTIN (SEQ ID NO: 1) (CDR1)

LFNPYNGGTT (SEQ ID NO:2) (CDR2)

ARRYYGYDAMDY(SEQ ID NO:3)(CDR3).

In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a light chain comprising one or more light chain CDRs selected from:

KSSQSVLYSSNQKNHLA(SEQ ID NO:4)(CDR1)

WASTRES (SEQ ID NO:5) (CDR2)

HQYLSSYT (SEQ ID NO:6) (CDR3).

In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising one or more heavy chain CDRs selected from:

GFNIKDYYMH (SEQ ID NO:7) (CDR1)

WIDPENDNTIY(SEQ ID NO:8)(CDR2)

ARDFGYVAWLVY(SEQ ID NO:9)(CDR3).

In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a light chain comprising one or more light chain CDRs selected from:

KASQNVDTAVA(SEQ ID NO:10)(CDR1)

SASNRYT (SEQ ID NO: 11) (CDR2)

QQYSSYPT (SEQ ID NO: 12) (CDR3).

In some embodiments, the isolated antibodies or antigen-binding fragments thereof or antibodies or fragments thereof provided herein are chimeric or humanized.

In some embodiments, the isolated antibody or antigen-binding fragment thereof or antibody or fragment thereof provided by the present invention is selected from the group consisting of a monoclonal antibody, scFv, Fab fragment, Fab' fragment and F(ab)' fragment. It should be noted that although scFv is not an antibody fragment in the strict sense, but a fusion protein, the antibody fragment of the present invention includes scFv, unless otherwise specified.

The present invention provides a nucleic acid encoding an antibody heavy chain comprising one or more heavy chain CDRs selected from the group consisting of: GYSFTGYTIN (SEQ ID NO: 1) (CDR1);

LFNPYNGGTT(SEQ ID NO:2)(CDR2);

ARRYYGYDAMDY(SEQ ID NO:3)(CDR3).

The present invention provides a nucleic acid encoding an antibody heavy chain comprising one or more heavy chain CDRs selected from the group consisting of: GFNIKDYYMH (SEQ ID NO: 7) (CDR1);

WIDPENDNTIY(SEQ ID NO:8)(CDR2);

ARDFGYVAWLVY(SEQ ID NO:9)(CDR3).

The present invention provides a nucleic acid encoding an antibody light chain comprising one or more light chain CDRs selected from the group consisting of: KSSQSVLYSSNQKNHLA (SEQ ID NO: 4) (CDR1);

WASTRES (SEQ ID NO: 5) (CDR2);

HQYLSSYT (SEQ ID NO:6) (CDR3).

The present invention provides a nucleic acid encoding an antibody light chain comprising one or more light chain CDRs selected from the group consisting of: KASQNVDTAVA (SEQ ID NO: 10) (CDR1);

SASNRYT (SEQ ID NO: 11) (CDR2)

QQYSSYPT (SEQ ID NO: 12) (CDR3).

The invention provides a host cell comprising one or more nucleic acids of the invention.

The invention provides an antibody or fragment thereof of the invention linked or conjugated to a therapeutic agent.

In some embodiments, the therapeutic agent is a cytotoxic agent, a radioisotope, an immunomodulatory agent, or a secondary antibody.

The present invention provides a method of inhibiting human Tim-3 in a subject, the method comprising administering the antibody or fragment thereof or human Tim-3 binding fragment thereof of the present invention in an amount effective to inhibit human Tim-3.

In some embodiments, the subject has cancer.

The present invention provides a method for inhibiting Tim-3-mediated T cell inhibition in a subject, the method comprising administering an antibody or fragment thereof of the present invention or a human Tim-3 binding fragment thereof in an amount effective to inhibit Tim-3-mediated T cell inhibition. The present invention provides a method for treating cancer in a subject, the method comprising administering an antibody or fragment thereof of the present invention in an amount effective to treat cancer in the subject.

In some embodiments, the cancer is a human Tim-3-positive cancer.

The present invention provides a method for detecting human Tim-3-positive cells in a subject, the method comprising administering an antibody or fragment thereof of the present invention conjugated to a detectable marker in an amount effective to label human Tim-3-positive cells, and then detecting human Tim-3-positive cells in the subject by detecting the presence of the marker in the subject.

In some embodiments, the marker is detected by imaging.

In some embodiments, the cell is a cancer cell.

In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is lung cancer, gastric cancer, head and neck cancer, schwannoma, melanoma, or follicular B-cell non-Hodgkin's lymphoma.

In some embodiments, the cancer comprises a solid tumor.

In some embodiments, the subject is receiving anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy.

In some embodiments, the anti-Tim-3 IgV domain antibody or its Tim-3 IgV domain binding fragment provided by the present invention comprises (i) a VH framework region comprising the human germline framework region sequence IGHV1-2*02, IGHV1-2*04, IGHV1-2*05, IGHV1-18*04, IGHV1-69-2*01, IGHV1-46*01, IGHD5-12*01, IGHD5-24*01, IGHD6-25*01, IGHJ3*01, IGHJ4*01. , IGHJ4*03, IGHJ6*01, IGHJ6*02 and/or (ii) a framework region of VL comprising the human germline framework region sequence IGKV1-13*02, IGKV1-27*01, IGKV3-7*02, IGKV4-1*01, IGKV1D-13*02, IGKV3D-7*01, IGKJ1*01, IGKJ2*01, IGKJ4*01, IGKJ4*02.

In some embodiments, the anti-Tim-3 IgV domain antibodies or Tim-3 IgV domain binding fragments thereof provided herein bind to the Tim-3 IgV domain with a binding affinity (KD) of about 1×10 ⁻⁹ M to about 1×10 ⁻¹² M.

In some embodiments, the anti-Tim-3 IgV domain antibodies or Tim-3 IgV domain binding fragments thereof provided by the present invention are monoclonal antibodies.

In some embodiments, the anti-Tim-3 IgV domain antibodies or Tim-3 IgV domain binding fragments thereof provided by the present invention are recombinant antibodies.

In some embodiments, the anti-Tim-3 IgV domain antibodies or Tim-3 IgV domain binding fragments thereof provided herein comprise human framework regions.

In some embodiments, the anti-Tim-3 IgV domain antibodies or Tim-3 IgV domain binding fragments thereof provided herein comprise a human constant region.

In some embodiments, the present invention provides anti-Tim-3 IgV domain antibodies.

In some embodiments, the invention provides Tim-3 IgV domain binding fragments of antibodies.

In some embodiments, the Tim-3 IgV domain binding fragment is a Fab, F(ab)2, or scFv.

The present invention provides an isolated nucleic acid molecule encoding an anti-Tim-3 IgV domain antibody or a Tim-3 IgV domain binding fragment thereof of the present invention. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is cDNA. In one embodiment, the nucleic acid is RNA.

The present invention provides a vector comprising the nucleic acid molecule of the present invention. The present invention also provides a host cell comprising the nucleic acid molecule of the present invention, or comprising the vector of the present invention.

The present invention provides a method for preparing an anti-Tim-3 IgV domain antibody or a Tim-3 IgV domain binding fragment thereof, the method comprising culturing a host cell under conditions enabling the host cell to produce the anti-Tim-3 IgV domain antibody or the Tim-3 IgV domain binding fragment thereof.

The present invention provides a composition comprising an anti-Tim-3 IgV domain antibody or a Tim-3 IgV domain binding fragment thereof of the present invention, and a carrier. In one embodiment, the composition is a pharmaceutical composition, and the carrier is a pharmaceutical carrier. The present invention also provides a pharmaceutical composition comprising the antibody or its binding fragment of the present invention and a pharmaceutically acceptable excipient.

The term "antibody" as used herein refers to a complete antibody, such as an antibody with a complete Fc region and Fv region. "Fragment" refers to any portion of an antibody or antibody portions linked together, such as Fab, F(ab)2, single-chain Fv (scFv), which is smaller than a complete antibody, but it is an antigen binding fragment and competes with a complete antibody with a specific binding fragment. In the present invention, the antigen is the human Tim-3 IgV domain.

For example, antibody fragments can be prepared by cutting whole antibodies or recombinant techniques, generally referring to Fundamental Immunology (Fundamental.Immunology Chapter 7 Paul, W. Editor, 2nd Edition, Raven Press, N.Y. (1989), incorporated herein by reference in its entirety). Antigen binding fragments can be prepared by DNA recombinant technology, or by enzymatic or chemical cleavage of whole antibodies, or by molecular biology techniques. In some embodiments, antibody fragments are Fab, Fab', F(ab')2, Fd, Fv, complementary determining region (CDR) fragments or single-chain antibodies (scFv, light chain variable domain (VL) and heavy chain variable domain (VH) connected by connecting peptides). In one embodiment, scFv comprises variable domain framework region sequences identical to human variable domain FR1, FR2, FR3 or FR4 sequences. In one embodiment, scFv comprises connecting peptides having a length of 5 to 30 amino acid residues. In one embodiment, scFv comprises connecting peptides comprising one or more of glycine, serine and threonine residues.

In one embodiment, the length of the connecting peptide of the scFv is 10-25 amino acids. In one embodiment, the connecting peptide comprises glycine, serine and/or threonine residues (see Bird 1988 and Huston 1988, both of which are incorporated herein by reference in their entirety), or comprises a polypeptide containing at least a portion of an antibody, a portion of which is sufficient to bind a Tim-3 IgV domain-specific antigen to the polypeptide, including bispecific antibodies. From N-terminus to C-terminus, the mature light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR, CDR3 and FR4 domains. The amino acid distribution of each domain is performed according to the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk 1987 or Chothia 1989, the contents of which are each incorporated herein by reference in their entirety. The term "polypeptide" as used herein encompasses polypeptide analogs of natural or artificial proteins, protein fragments, and protein sequences. Polypeptides can be in monomeric or multimeric form. As used herein, an Fd fragment is an antibody fragment consisting of a VH and CH1 domain; an Fv fragment consists of a single-arm VL and VH domain of an antibody; and a dAb fragment (Ward 1989, incorporated herein by reference in its entirety) consists of a VH domain. In some embodiments, the fragment is at least 5, 6, 8, or 10 amino acids long. In other embodiments, the fragment is at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150, or 200 amino acids long.

The term "monoclonal antibody" as used herein refers to an antibody in a substantially homogeneous antibody population, that is, except for a small amount of natural mutations that may exist, the individual antibodies in the antibody population are all the same. Therefore, the modifier "monoclonal" indicates that the nature of the antibody is not a mixture of discrete antibodies. In certain embodiments, the monoclonal antibody includes an antibody having a sequence that binds to a human Tim-3 IgV domain polypeptide. Polyclonal antibodies typically contain a variety of antibodies against multiple determinants (epitopes), while monoclonal antibodies are different in that each monoclonal antibody is only directed against a single antigenic determinant. In addition to its specificity, the advantage of monoclonal antibodies is that they are generally not contaminated by other immunoglobulins. Therefore, monoclonal antibodies of known sequences can be obtained by non-hybridoma methods such as suitable recombinant techniques.

In one embodiment, the antibody is isolated. The term "isolated antibody" as used herein means that it is derived from or derived from an antibody that has one, two, three, or four of the following: (1) is not associated with its naturally associated components that accompany it in its native state, (2) does not contain other proteins from the same species, (3) is expressed by a cell from a different species, and (4) does not occur in nature.

In one embodiment, the antibody is humanized. The "humanized" form of a non-human (such as mouse) antibody is a chimeric antibody containing a minimum of non-human immunoglobulin sequences. In one embodiment, a humanized antibody is a human immunoglobulin (receptor antibody) in which the residues of a hypervariable region (HVR or CDR) are replaced by residues of a HVR (or CDR) having the desired specificity, affinity and/or ability in a non-human species (donor antibody) such as a mouse, rat, rabbit or non-human primate. In one embodiment, the antibody has 1, 2, 3, 4, 5 or 6 of the heavy and light chain CDR1-3 of the mouse antibody (mAb 15B4 and mAb 50B5) described in the present invention. In a preferred embodiment, the residues of the framework region (FR) of the mouse monoclonal antibody are replaced by residues of the framework region (FR) of the corresponding human immunoglobulin variable domain. These modifications can be made in the embodiment to further improve the antibody performance. In addition, in a specific embodiment, the humanized antibody may include residues not found in the receptor antibody or the donor antibody. In one embodiment, a humanized antibody does not comprise residues that are not found in the recipient antibody or the donor antibody. In general, a humanized antibody comprises substantially all variable domains having at least one, usually two, of the following, wherein all or substantially all of the hypervariable coiled regions correspond to the hypervariable coiled regions of a non-human immunoglobulin (e.g., mAb 15B4 and mAb 50B5 described herein), and all or substantially all of the FRs are FRs in human immunoglobulin sequences. A humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin, see, e.g., Jones 1986; Riechmann 1988; Presta 1992; Vaswani & Hamilton 1998; Harris 1995; Hurle & Gross 1994 and U.S. Pat. Nos. 6,982,321 and 7,087,409, the contents of each of which are incorporated herein by reference in their entirety. In one embodiment, the humanized antibody comprises residues not found in the recipient antibody or the donor antibody, such as the modified antibody Fc region described in WO99/58572, the contents of which are incorporated herein by reference in their entirety. Humanization techniques for monoclonal antibodies are well known, such as described in U.S. Patents 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370, the contents of which are each incorporated herein by reference in their entirety. Many "humanized" antibody molecules containing antigen-binding sites derived from non-human immunoglobulins have been described, including antibodies having rodent or modified rodent V regions and their associated CDRs fused to human constant domains, see, e.g., Winter 1991, Lobuglio 1989, Shaw 1987, and Brown 1987, the contents of which are each incorporated herein by reference in their entirety. Other references describe grafting rodent hypervariable regions or CDRs into human supporting framework regions (FRs) prior to fusion with appropriate human antibody constant domains, see, e.g., Riechmann 1988, Verhoeyen 1988 and Jones 1986, the contents of which are each incorporated herein by reference in their entirety. Another reference describes rodent CDRs supported by recombinantly engineered rodent framework regions, see European Patent Publication No. 0519596, incorporated herein by reference in its entirety. These "humanized" molecules are designed to minimize the undesirable immune response in rodent anti-human antibody molecules, which limits the duration and effectiveness of these antibodies in human therapeutic applications. The constant region of an antibody is genetically engineered to be immunologically inert (e.g., not induce complement lysis), see, e.g., PCT Publication No. WO99/58572; British Patent Application No. 9809951.8. Other methods of humanizing antibodies that may be utilized are disclosed by Daugherty 1991; U.S. Pat. Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; 6,350,861 and PCT Publication No. WO01/27160, the contents of each of which are incorporated herein by reference in their entirety.

Other forms of humanized antibodies, wherein one or more or all CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 or CDR H3) are altered relative to the original antibody, are also referred to as one or more CDRs "derived" from one or more CDRs of the original antibody. In one embodiment, the antibodies provided herein are humanized mAb15B4 and mAb 50B5, wherein one or more or all CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 or CDR H3) are altered relative to the original antibody. In one embodiment, the antibodies provided herein are humanized mAb15B4 and mAb 50B5, whose CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 or CDR H3) are not altered relative to the original antibody.

In some embodiments, antibodies or fragments can be produced by recombinant technology, for example, expressing antibodies using a recombinant expression vector transfected into a host cell, isolating antibodies from a recombinant human antibody combinatorial library, isolating antibodies from animals (e.g., mice) into which human immunoglobulin genes have been transferred.

In one embodiment, the anti-Tim-3 IgV domain antibody of the present invention is capable of specifically binding or specifically binding to a human Tim-3 IgV domain. As used herein, the term "capable of specific binding" or "specifically binding" refers to a dissociation constant of an antibody portion or fragment binding to a (specific) antigen <1 μM, preferably <1 nM, and most preferably <10 pM. In one embodiment, the antibody (or fragment) of the present invention has a Kd of less than 10.0 nM for the Tim-3 IgV domain. In one embodiment, the antibody (or fragment) of the present invention has a Kd of less than 1.0 nM for the Tim-3 IgV domain. In one embodiment, the antibody (or fragment) of the present invention has a Kd of less than 0.5 nM for the Tim-3 IgV domain. In one embodiment, the antibody (or fragment) of the present invention has a Kd of less than or equal to 0.1 nM for the Tim-3 IgV domain.

The term "Kd" as used herein refers to the dissociation constant of the antibody-antigen interaction. One method for determining the Kd or binding affinity of an antibody to the Tim-3 IgV domain is to detect the binding affinity of a monofunctional Fab fragment of the antibody. (The binding constant is the inverse dissociation constant). Monofunctional Fab fragments can be obtained by cleaving the antibody (e.g., IgG) with papain or recombinant expression. The affinity of the anti-human Tim-3 IgV domain antibody fragment can be determined, for example, by surface plasmon resonance (BIAcore3000 ^TM surface plasmon resonance (SPR) system, BlaAcore Inc., Piscataway NJ). The CM5 chip can be activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen is diluted with 10mM sodium acetate at pH 4.0 and injected into the activated chip at a concentration of 0.005mg/mL. Different flow times were used through each chip channel to obtain two sets of antigen density: 100-200 response units (RU) for detailed kinetic studies and 500-600RU for screening. The purified Fab samples were gradient diluted (0.1-10× estimated Kd) and injected at 100 μL/min for 1 minute, with a dissociation time of up to 2 hours. The concentration of Fab protein was determined by ELISA and/or SDS-PAGE electrophoresis using a known concentration of Fab (determined by amino acid analysis) as a standard. The BIA evaluation software was used to fit the data with a 1:1 Langmuir binding model, and the kinetic association rate (kon) and dissociation rate (koff) were obtained simultaneously (see Karlsson et al., Kinetic and concentration analysis using BIA technology, Methods: A Companion to Methods Enzymol 6: 99-110 (1994), the contents of which are incorporated herein by reference in their entirety), and the equilibrium dissociation constant (Kd) value was calculated by koff/kon. This protocol is applicable to determining the binding affinity of antibodies or fragments to any antigen and can also be determined using other protocols known in the art, such as ELISA.

"Specific binding" to an epitope of an antibody or polypeptide is a term well known in the art, and methods for determining such specific or preferential binding are also well known in the art. If a molecule reacts or binds to a specific cell or substance more frequently, more rapidly, more persistently and/or with a stronger affinity than its reaction and binding to other cells or substances, the molecule is considered to exhibit "specific binding" or "preferential binding". If the antibody binds to the target with a stronger affinity, affinity retention, more easily and/or more persistently than its binding to other substances, the antibody "specifically binds" or "preferentially binds" to the target. For example, an antibody that specifically binds or preferentially binds to the human Tim-3 IgV domain means that the antibody binds to the human Tim-3 IgV domain with a stronger affinity, affinity retention, faster and/or more persistently than binding to other epitopes of Tim-3 or non-Tim-3 epitopes. It should also be understood by reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily (although it may include) exclusive binding.

The term "competition" used herein for antibodies refers to a first antibody or its antigen-binding portion binding to an epitope in a sufficiently similar manner to a second antibody or its antigen-binding portion, and the binding of the first antibody to its relevant epitope is detectably reduced when the second antibody is present, compared to the absence of the second antibody. Alternatively, it may be the case (but not necessarily) that the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody. That is, the first antibody is able to inhibit the binding of the second antibody to its epitope, while the second antibody is unable to inhibit the binding of the first antibody to its epitope. However, when each antibody detectably inhibits the binding of another antibody to its relevant epitope or ligand, whether to the same, higher or lower degree, it is considered that the antibodies "cross-compete" with each other to bind to their respective epitopes. Competitive and cross-competitive antibodies are included in the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational changes, or binding to a common epitope or portion thereof, etc.), skilled technicians should know that such competitive and/or cross-competitive antibodies are included and applicable to the methods disclosed in the present invention according to the teachings provided herein.

According to the amino acid sequence of the constant domain of the heavy chain of the antibody, antibodies (immunoglobulins) can be divided into different categories. For example, the antibody or fragment can be any one of IgG, IgD, IgE, IgA or IgM antibodies or their fragments. In one embodiment, the antibody is immunoglobulin G. In one embodiment, the antibody fragment is a fragment of immunoglobulin G. In one embodiment, the antibody is IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4. In one embodiment, the antibody comprises a sequence from human IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3 or human IgG4. Combinations of any of the above-mentioned antibody subtypes can also be used. The antibody serum half-life should be considered when selecting the required antibody type. For example, the serum half-life of IgG is generally 23 days, the serum half-life of IgA is 6 days, the serum half-life of IgM is 5 days, the serum half-life of IgD is 3 days, and the serum half-life of IgE is 2 days (Abbas AK, Lichtman AH, Pober JS. Cellular and Molecular Immunology, 4th Edition, W.B. Saunders Co., Philadelphia, 2000, herein incorporated by reference in its entirety).

The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy or light chain of an antibody. The variable domain of the heavy chain may be referred to as "VH" and the variable domain of the light chain may be referred to as "VL". These domains contain the antigen binding site and are generally the most variable parts of an antibody. The term "variable" refers to the fact that the sequence of certain parts of the variable domain varies greatly between antibodies and is used for the binding of each specific antibody to its specific antigen and its specificity. However, this variability is not evenly distributed within the variable domain of an antibody, but is mainly concentrated in three segments called hypervariable regions (HVRs or CDRs) within the light and heavy chain variable domains. The more conserved parts of the variable domain are called framework regions (FRs). Each natural heavy and light chain variable domain each contains four FR regions that are primarily in a β-sheet configuration, which are connected together by three CDRs to form a coiled connection and, in some cases, form part of a β-sheet configuration. The CDRs of each chain are held together in close proximity by the FRs and combined with the CDRs of the other chain to form the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in the binding of antibodies to antigens, but exhibit a variety of effector functions, such as the role of antibodies in antibody-dependent cellular toxicity.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

"Framework region" or "FR" residues refer to those residues in the variable domain other than the HVR residues defined herein. The term "hypervariable region" or "HVR" as used herein refers to a region in an antibody variable domain where the sequence is highly variable and/or forms a structurally defined coil. Typically, antibodies contain six hypervariable regions: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 are the most diverse regions of the six HVRs, and H3 in particular is believed to play a unique role in conferring precise specificity to antibodies, see, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains (see, e.g., Hamers-Casterman 1993; Sheriff & Constantine 1996). Multiple HVR descriptions are in use and are covered herein. Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), incorporated herein by reference as a whole). The heavy chain and light chain each have CDRs 1, 2, and 3. Chothia refers to the position of the structural curl (Chothia & Lesk 1987). AbM HVRs represent a compromise between the Kabat HVR and Chothia structural curl regions and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVR is based on an analysis of available composite crystal structures. An HVR may comprise "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al., supra.

The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from the position of the amino acid residue of Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine in the Fc region can be removed, for example, during the preparation or purification of the antibody, or removed by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Therefore, the intact antibody used herein can be an antibody with or without a C-terminal lysine.

The composition or pharmaceutical composition comprising the antibody, scFv or antibody fragment of the present invention preferably comprises a stabilizer to prevent the loss of activity or structural integrity due to denaturation, oxidation or aggregation during storage and transportation of the protein before use. The composition or pharmaceutical composition may comprise any combination of one or more of salts, surfactants, pH regulators and tension regulators (e.g., sugars) to overcome the problem of aggregation. When the composition or pharmaceutical composition is used as an injection, the desired pH value is approximately in the neutral pH range, and the surfactant content is minimized to avoid bubbles in the formulation that are not conducive to injection into the subject. In one embodiment, the composition or pharmaceutical composition is in liquid form, capable of maintaining the stability of a high concentration of biologically active antibodies in the solution, and is suitable for inhalation or parenteral administration. In one embodiment, the composition or pharmaceutical composition is suitable for intravenous, intramuscular, intraperitoneal, intradermal and/or subcutaneous injection. In one embodiment, the composition or pharmaceutical composition is in liquid form, and the risk of producing bubbles and anaphylactoid side effects has been minimized. In one embodiment, the composition or pharmaceutical composition is isotonic. In one embodiment, the pH value of the composition or pharmaceutical composition is 6.8 to 7.4.

In one embodiment, the ScFvs or antibody fragments disclosed herein are lyophilized and/or freeze-dried and reconstituted for use.

Examples of pharmaceutically acceptable carriers include, but are not limited to, phosphate buffered saline, sterile water (including water for injection USP), emulsions (e.g., oil/water emulsions), and various types of wetting agents. The diluent for aerosol or parenteral administration is preferably phosphate buffered saline or physiological saline (0.9%), such as 0.9% sodium chloride solution, USP. Compositions containing such carriers are prepared by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990 and Remington, The Science and Practice of Pharmacy 20th edition. Mack Publishing, 2000, the contents of which are each incorporated herein by reference in their entirety). In non-limiting examples, one or more of disodium hydrogen phosphate, potassium chloride, potassium dihydrogen phosphate, polysorbate 80 (such as 2-[2-[3,5-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl(E)-octadec-9-enoate), disodium salt of ethylenediaminetetraacetic acid, sucrose, sodium dihydrogen phosphate monohydrate, and sodium dihydrogen phosphate dihydrate may be included.

The antibodies or antibody fragments, or compositions or pharmaceutical compositions of the present invention may be provided in lyophilized or any other suitable form, including but not limited to injectable or inhalable solutions, gels and tablets.

The term "Fc domain" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which includes a native sequence Fc region and a variant Fc region. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from the position of the amino acid residue of Cys226 or Pro230 to its carboxyl terminus. The lysine at the C-terminus of the Fc region may be removed, for example, by recombinant engineering of the nucleic acid encoding it. In some embodiments, the antibody comprises an Fc domain. In one embodiment, the sequence of the Fc domain is identical to or has a sequence similarity of 99% or more to that of a human IgG1 Fc domain. In one embodiment, the sequence of the Fc domain is identical to or has a sequence similarity of 99% or more to that of a human IgG2 Fc domain. In one embodiment, the sequence of the Fc domain is identical to or has a sequence similarity of 99% or more to that of a human IgG3 Fc domain. In one embodiment, the sequence of the Fc domain is identical to or has a sequence similarity of 99% or more to that of a human IgG4 Fc domain. In one embodiment, the Fc domain has no mutations. In one embodiment, the CH2-CH3 domain interface of the Fc domain is mutated to increase the affinity of IgG for FcRn under acidic rather than neutral pH conditions (Dall'Acqua 2006; Yeung 2009). In one embodiment, the Fc domain has the same sequence as the human IgG1 Fc domain.

In some embodiments, the present invention encompasses modified forms for variable regions. For example, the present invention includes antibodies with functionally equivalent variable regions and CDRs that do not significantly affect their performance, as well as variants with enhanced or reduced activity and/or affinity. For example, the amino acid sequence of an antibody can be mutated to obtain an antibody with the desired binding affinity to human Tim-3 IgV. The modification of polypeptides is a routine operation in the art and does not need to be described in detail here. Examples of modified polypeptides include polypeptides with conservative amino acid residue substitutions, one or more amino acid deletions or additions (which do not significantly and harmfully change the functional activity, or mature (enhance) the affinity of the polypeptide to its ligand), or application of chemical analogs.

Amino acid sequence insertions include fusions of 1 residue to 100 or more residues at the amino and/or carboxyl termini, as well as insertions of single or multiple amino acid residues within the sequence. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertion variants of antibody molecules include fusions to enzymes or polypeptides at the N or C termini of the antibody to increase the serum half-life of the antibody.

Substitution variants are at least one amino acid residue in the antibody molecule that is removed and a different residue is inserted in that position. The most significant sites for substitution mutagenesis include hypervariable regions, but changes in framework regions are also considered. Conservative substitutions are shown in Table 1 with the heading "Conservative Substitutions". If such substitutions result in changes in biological activity, more substantial changes named "Exemplary Substitutions" in Table 1 or further described below with reference to amino acid classes may be introduced and the product screened.

Table 1: Amino Acid Substitutions

Substantial modification of the biological properties of antibodies can be achieved by selective substitution, i.e., selecting residues that differ significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the substituted region, such as β-sheet or helical conformation; (b) the charge or hydrophobicity at the target site of the molecule; or (c) the bulk of the side chain. Based on common side chain properties, natural residues can be divided into the following categories:

(1) Non-polar: norleucine, Met, Ala, Val, Leu, Ile;

(2) Polarity without charge: Cys, Ser, Thr, Asn, Gln;

(3) Acidic (negatively charged): Asp, Glu;

(4) Basic (positively charged): Lys, Arg;

(5) Residues that affect side chain orientation: Gly, Pro; and

(6) Aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions may be made by exchanging a member of one of these classes for another.

A type of substitution, for example, one or more cysteines in an antibody that may be chemically reactive are changed to another residue, such as, but not limited to, alanine or serine. For example, a non-classical cysteine can be substituted. Replacements can be made in the CDR region or framework region of the variable domain or the constant region of the antibody. In some embodiments, the cysteine is a classical cysteine. Serine can also be used to replace any cysteine residue that does not participate in maintaining the correct configuration of the antibody to improve the oxidative stability of the molecule and prevent abnormal cross-linking. On the contrary, cysteine can be added to the antibody to improve its stability, especially when the antibody is an antibody fragment such as an Fv fragment.

The antibody can also be modified in, for example, the variable domain of the heavy chain and/or light chain to change the binding properties of the antibody. Changes in the variable domain can change the binding affinity and/or specificity. In some embodiments, the conservative amino acid substitutions made in the CDR domain do not exceed 1-5. In other embodiments, the conservative amino acid substitutions made in the CDR domain do not exceed 1-3. For example, mutations can be made in one or more CDR regions to increase or decrease the KD, or k _off , of the antibody to the human Tim-3 IgV domain, or to change the binding specificity of the antibody. Site-directed mutagenesis techniques are well known in the art.

Modifications or mutations may also be made in the framework region or constant region to increase the half-life of the anti-human Tim-3 IgV domain antibody, see, for example, PCT Publication No. WO 00/09560. Mutations may also be made in the framework region or constant region to alter the immunogenicity of the antibody, provide a site for covalent or non-covalent binding to another molecule, or alter properties such as complement fixation, FcR binding, and antibody-dependent cell-mediated cytotoxicity. According to the present invention, an antibody may have mutations in one or more of the CDRs or framework regions or constant regions of the variable domain.

In one embodiment, the antibodies of the invention are recombinantly produced. In one embodiment, the fusion protein is produced in a eukaryotic expression system.

In one embodiment, the fusion protein produced by the eukaryotic expression system comprises a glycosylation modification at the residue corresponding to Asn297 of the Fc portion.

In one embodiment, a composition or pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention is substantially pure with respect to the antibody or antigen-binding fragment thereof. A composition or pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention is "substantially pure" with respect to the antibody or antigen-binding fragment thereof when at least 60% to 75% of the sample is a single species of antibody or antigen-binding fragment thereof. A substantially pure composition or pharmaceutical composition (comprising an antibody or antigen-binding fragment thereof of the invention) is one in which the portion that is an antibody or antigen-binding fragment contains 60%, 70%, 80% or 90%, preferably about 95%, and more preferably more than 99% of a single species of antibody or antigen-binding fragment thereof. Purity or homogeneity can be determined by a variety of means well known in the art, such as polyacrylamide gel electrophoresis or HPLC.

In one embodiment, human Tim3 has the following sequence:

MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGA

CPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQI

PGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDIN

LTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEK

IQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP(SEQ ID NO:13)

In one embodiment, the sequence of the IgV domain of the antibody comprises: SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVN YWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPA (SEQ ID NO: 22)

As used herein, the term "and/or", for example option A and/or option B, includes (i) option A alone, (ii) option B alone, and (iii) option A plus option B.

All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or clearly contradicted by context.

In the following description, numerous experimental details are set forth to provide a better understanding of the present invention.

Example

Production of antibodies that specifically bind to the human Tim-3 IgV domain: The coding region of the human Tim-3 IgV gene (S22-A132) was fused to the human IgG1 Fc tag in the pMT/BiP plasmid to obtain the Tim-3 IgV-Ig fusion protein (Zhao 2013). The fusion protein was expressed in the S2 system and then purified. Mice were immunized with the Tim-3 IgV-Ig fusion protein, and spleen cells and myeloma cells NSO were fused using standard techniques to produce hybridomas.

Identification of mAb 50B5: mAb 50B5 was obtained, which has an IgG1 heavy chain and a kappa light chain. ELISA analysis showed that mAb 50B5 was able to bind to Tim-3-IgV protein and the full-length extracellular region protein of Tim-3 (IgV-mucin-stalk domain), but not to human IgG protein (Figure 1). FACS analysis showed that mAb 50B5 was able to bind to human Tim-3 expressed on cell lines, but not to mouse Tim-3 expressed on cell lines (Figure 2).

The binding affinity of mAb 50B5 to human Tim-3 protein was measured to be 0.13 nM KD by surface plasmon resonance (Figure 3). In order to determine the blocking function of mAb 50B5, a binding assay was performed and it was found that mAb 50B5 was able to block the binding of human Tim-3 to phosphatidylserine expressed on dexamethasone-treated Jurkat T cells. Tim-3 is an inhibitory receptor expressed by T cells that produce IFN-g, so a mixed lymphocyte reaction was used to detect the antagonistic ability of mAb 50B5. Mature dendritic cells differentiated from monocytes in donor PBMC were incubated with purified T cells from another donor PBMC for four days. It was found that Tim-3 was expressed on the surface of split CD4 T cells and split CD8 T cells, but not on the surface of unsplit CD4 T cells and unsplit CD8 T cells (Figure 4A). In the above mixed lymphocyte reaction, mAb 50B5 and mouse IgG1 control were added respectively. The results showed that mAb50B5 increased IFN-g production by more than three times that of the IgG1 control (Figure 4B).

Taken together, these results indicate that mAb 50B5 is an antagonist antibody against human Tim-3. Finally, the mAb50B5 hybridoma was sequenced and found to have a single VH and VL sequence:

50B5 heavy chain: DNA sequence (414 bp)

Leader peptide sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

(Leader peptide sequence is shown in bold, CDRs 1-3 are underlined)

50B5 heavy chain: amino acid sequence (138aa)

Leader peptide sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

(Leader peptide sequence is shown in bold, CDRs 1-3 are underlined)

50B5 light chain: DNA sequence (396 bp)

Leader peptide sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

(Leader peptide sequence is shown in bold, CDRs 1-3 are underlined)

50B5 light chain: amino acid sequence (132aa)

Leader peptide sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

(Leader peptide sequence in bold, CDRs 1-3 underlined)

Identification of mAb 15B4: mAb 15B4 is another antibody generated by the techniques described above. mAb 15B4 has an IgG1 heavy chain and a kappa light chain, and FACS analysis showed that it can bind to human Tim-3 expressed on the cell line (Figure 5). By surface plasmon resonance, the binding affinity of mAb 15B4 to human Tim-3 protein was measured to be 0.32nM KD (Figure 6). In order to determine the blocking function of mAb 50B5, a binding assay was performed and it was found that mAb 15B4 was able to block the binding of human Tim-3 to phosphatidylserine expressed on dexamethasone-treated Jurkat T cells. Finally, the 15B4 hybridoma was sequenced and found to have a single VH and VL sequence:

15B4 heavy chain: DNA sequence (414 bp)

Leader peptide sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

(Leader peptide sequence in bold, CDRs 1-3 underlined)

15B4 heavy chain: amino acid sequence (138aa)

Leader peptide sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

(Leader peptide sequence in bold, CDRs 1-3 underlined)

15B4 light chain: DNA sequence (378 bp)

Leader peptide sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

(Leader peptide sequence in bold, CDRs 1-3 underlined)

15B4 light chain: amino acid sequence (126aa)

Leader peptide sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

(Leader peptide sequence in bold, CDRs 1-3 underlined)

MSQSQVFVFAFLWLSGVDGDIVMTQSQEFMSTSVGDRVSITC KASQNVDTAVA WY

QQKPGQSPKLLIY SASNRYT GVPDRFTGTGSGTDFTLTINNMQSEDLADYFC QQYS

SYPT FGGRTKLEIKR

(SEQ ID NO:21).

References

Bird et al.Science 242(4877):423-426(1988)

Brown et al. Cancer Res 47(13):3577-3583(1987)

Cao et al. Immunity 26(3):311-321(2007)

Chiba et al. Nat Immunol 13(9):832-842(2012)

Chothia&Lesk J Mol Biol 196(4):901-917(1987)

Chothia et al.Nature 342(6252):877-883(1989)

Dall’Acqua et al.J Biol Chem 281(33):23514-23524(2006)

Daugherty et al. Nucleic Acids Res 19(9):2471-2476(1991)

DeKruyff et al.J Immunol 184(4):1918-1930(2010)

Hamers-Casterman et al. Nature 363(6428):446-448(1993)

Harris Biochem Soc Trans 23(4):1035-1038(1995)

Hurle&Gross Curr Opin Biotechnol 5(4):428-433(1994)

Huston et al. Proc Natl Acad Sci USA 85(16):5879-5883(1988)

Jones et al.Nature 321(6069):522-525(1986)

Koyama et al. Nat Commun 7:10501(2016)

Lobuglio et al. Proc Natl Acad Sci USA 86(11):4220-4224(1989)

Monney et al.Nature 415(6871):536-541(2002)

Presta Curr Opin Biotechnol 3(4):394-398(1992)

Riechmann et al.Nature 332(6162):323-327(1988)

Shaw et al.J Immunol 138(12):4534-4538(1987)

Sheriff&Constantine Nat Struct Biol 3(9):733-736(1996)

Vaswani&Hamilton Ann Allergy Asthma Immunol 81(2):105-115(1998)

Verhoeyen et al.Science 239(4847):1534-1536(1988)

Ward et al.Nature 341(6242):544-546(1989)

Wilker et al. Int Immunol 19(6):763-773(2007)

Winter et al.Nature 349(6307):293-299(1991)

Yeung et al.J Immunol 182:7663-7671(2009)

Zhao et al. Proc Natl Acad Sci USA 110(24):9879-9884(2013)

Zhu et al.Nat Immunol 6(12):1245-1252(2005)

Claims (33)

1. An antibody or antigen-binding fragment thereof that binds to an IgV domain of human Tim-3 (T cell immunoglobulin and mucin-3) or an IgV-mucin-stem domain of human Tim-3, comprising:

a) A heavy chain comprising one or more heavy chain CDRs selected from the group consisting of:

GYSFTGYTIN(SEQ ID NO:1)(CDR1)；

LFNPYNGGTT(SEQ ID NO:2)(CDR2)；

ARRYYGYDAMDY(SEQ ID NO:3)(CDR3)；

Or (b)

GFNIKDYYMH(SEQ ID NO:7)(CDR1)；

WIDPENDNTIY(SEQ ID NO:8)(CDR2)；

ARDFGYVAWLVY (SEQ ID NO: 9) (CDR 3); and

B) A light chain comprising one or more light chain CDRs selected from the group consisting of:

KSSQSVLYSSNQKNHLA(SEQ ID NO:4)(CDR1)；

WASTRES(SEQ ID NO:5)(CDR2)；

HQYLSSYT(SEQ ID NO:6)(CDR3)；

Or (b)

KASQNVDTAVA(SEQ ID NO:10)(CDR1)；

SASNRYT(SEQ ID NO:11)(CDR2)；

QQYSSYPT(SEQ ID NO:12)(CDR3)。

2. An antibody or antigen-binding fragment thereof that binds to an IgV domain of human Tim-3 or an IgV-mucin-stem domain of human Tim-3, comprising:

a heavy chain comprising the following heavy chain CDRs:

GYSFTGYTIN(SEQ ID NO:1)(CDR1)；

LFNPYNGGTT(SEQ ID NO:2)(CDR2)；

ARRYYGYDAMDY (SEQ ID NO: 3) (CDR 3); and

A light chain comprising the following light chain CDRs:

KSSQSVLYSSNQKNHLA(SEQ ID NO:4)(CDR1)；

WASTRES(SEQ ID NO:5)(CDR2)；

HQYLSSYT(SEQ ID NO:6)(CDR3)。

3. An antibody or antigen-binding fragment thereof that binds to an IgV domain of human Tim-3 or an IgV-mucin-stem domain of human Tim-3, comprising:

a heavy chain comprising the following heavy chain CDRs:

GFNIKDYYMH(SEQ ID NO:7)(CDR1)；

WIDPENDNTIY(SEQ ID NO:8)(CDR2)；

ARDFGYVAWLVY (SEQ ID NO: 9) (CDR 3); and

A light chain comprising the following light chain CDRs:

KASQNVDTAVA(SEQ ID NO:10)(CDR1)；

SASNRYT(SEQ ID NO:11)(CDR2)；

QQYSSYPT(SEQ ID NO:12)(CDR3)。

4. The antibody or fragment thereof of any one of claims 1-3, wherein the framework regions of the light and heavy chains are human framework regions, or have at least 85% identity to human framework regions.

5. The antibody or fragment thereof of claim 4, wherein the framework regions of the light and heavy chains are human framework regions.

6. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds with an affinity of 10.0nM KD or greater to an IgV domain of human Tim-3 or an IgV-mucin-stem domain of human Tim-3.

7. The isolated antibody or antigen-binding fragment thereof of claim 6, wherein the antibody or antigen-binding fragment thereof binds with an affinity of 0.5nM KD or greater to an IgV domain of human Tim-3 or an IgV-mucin-stem domain of human Tim-3.

8. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof has a human Fc region sequence.

9. The isolated antibody or antigen-binding fragment thereof of any one of claims 6, 7, or 8, wherein the antibody or antigen-binding fragment thereof inhibits binding of human Tim-3 to phosphatidylserine expressed on dexamethasone-treated Jurkat T cells.

10. The isolated antibody or antigen-binding fragment thereof of any one of claims 6-9, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising one or more heavy chain CDRs selected from the group consisting of:

GYSFTGYTIN(SEQ ID NO:1)(CDR1)

LFNPYNGGTT(SEQ ID NO:2)(CDR2)

ARRYYGYDAMDY(SEQ ID NO:3)(CDR3)。

11. The isolated antibody or antigen-binding fragment thereof of any one of claims 6-10, wherein the antibody or antigen-binding fragment thereof comprises a light chain comprising one or more light chain CDRs selected from the group consisting of:

KSSQSVLYSSNQKNHLA(SEQ ID NO:4)(CDR1)

WASTRES(SEQ ID NO:5)(CDR2)

HQYLSSYT(SEQ ID NO:6)(CDR3)。

12. The isolated antibody or antigen-binding fragment thereof of any one of claims 6-9, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising one or more heavy chain CDRs selected from the group consisting of:

GFNIKDYYMH(SEQ ID NO:7)(CDR1)

WIDPENDNTIY(SEQ ID NO:8)(CDR2)

ARDFGYVAWLVY(SEQ ID NO:9)(CDR3)。

13. The isolated antibody or antigen-binding fragment thereof of any one of claims 6-9 or 12, wherein the antibody or antigen-binding fragment thereof comprises a light chain comprising one or more light chain CDRs selected from the group consisting of:

KASQNVDTAVA(SEQ ID NO:10)(CDR1)

SASNRYT(SEQ ID NO:11)(CDR2)

QQYSSYPT(SEQ ID NO:12)(CDR3)。

14. the isolated antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the antibody or fragment thereof is chimeric or humanized.

15. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, scFv, fab fragment, fab 'fragment, and F (ab)' fragment.

16. A nucleic acid encoding an antibody heavy chain comprising one or more heavy chain CDRs selected from the group consisting of:

GYSFTGYTIN(SEQ ID NO:1)(CDR1)；

LFNPYNGGTT(SEQ ID NO:2)(CDR2)；

ARRYYGYDAMDY(SEQ ID NO:3)(CDR3)。

17. a nucleic acid encoding an antibody heavy chain comprising one or more heavy chain CDRs selected from the group consisting of:

GFNIKDYYMH(SEQ ID NO:7)(CDR1)；

WIDPENDNTIY(SEQ ID NO:8)(CDR2)；

ARDFGYVAWLVY(SEQ ID NO:9)(CDR3)。

18. A nucleic acid encoding an antibody light chain comprising one or more light chain CDRs selected from the group consisting of:

KSSQSVLYSSNQKNHLA(SEQ ID NO:4)(CDR1)；

WASTRES(SEQ ID NO:5)(CDR2)；

HQYLSSYT(SEQ ID NO:6)(CDR3)。

19. A nucleic acid encoding an antibody light chain comprising one or more light chain CDRs selected from the group consisting of:

KASQNVDTAVA(SEQ ID NO:10)(CDR1)；

SASNRYT(SEQ ID NO:11)(CDR2)

QQYSSYPT(SEQ ID NO:12)(CDR3)。

20. A host cell comprising one or more nucleic acids of any one of claims 14-17.

21. The antibody or fragment thereof of any one of claims 1-15, wherein the antibody or fragment thereof is linked or conjugated to a therapeutic agent.

22. The antibody or fragment thereof of claim 21, wherein the therapeutic agent is a cytotoxic agent, a radioisotope, an immunomodulatory agent, or a secondary antibody.

23. A method of inhibiting human Tim-3 in a subject, wherein the method comprises administering the antibody or fragment thereof of any one of claims 1-15, or a human Tim 3-binding fragment thereof, in an amount effective to inhibit human Tim-3.

24. The method of claim 23, wherein the subject has cancer.

25. A method of inhibiting Tim-3 mediated T cell inhibition in a subject, wherein the method comprises administering the antibody or fragment thereof of any one of claims 1-15, or a human Tim3 binding fragment thereof, in an amount effective to inhibit Tim-3 mediated T cell inhibition.

26. A method of treating cancer in a subject, wherein the method comprises administering the antibody or fragment thereof of any one of claims 1-15 in an amount effective to treat cancer in the subject.

27. The method of claim 26, wherein the cancer is a human Tim-3-positive cancer.

28. A method of detecting human Tim-3 positive cells in a subject, wherein the method comprises administering the antibody or fragment thereof of any one of claims 1-15 conjugated to a detectable label in an amount effective to label human Tim-3 positive cells, and then detecting human Tim-3 positive cells in the subject by detecting the presence of the label in the subject.

29. The method of claim 28, wherein the label is detected by imaging.

30. The method of claim 28 or 29, wherein the cell is a cancer cell.

31. The method of any one of claims 24, 26, 27 or 30, wherein the cancer is a hematological malignancy.

32. The method of any one of claims 24, 26, 27 or 30, wherein the cancer comprises a solid tumor.

33. The method of any one of claims 24, 26, 27, or 30-32, wherein the subject is receiving anti-PD-1, anti-PD-L1, or anti-CTLA 4 treatment.